The present invention relates to N-substituted 1-(2-butyrolactones and 2-thiobutyrolactones)-isoquinolines, their preparation, pharmaceutical compositions containing them and their use as stimulant of xcex3-aminobutyric acid activity and as medicament preferably intended for treating nervous disorders.
xcex3-aminobutyric acid (or GABA (I)) is the most important inhibiting neurotransmitter of the central nervous system. It acts at the level of three distinct classes of receptors called GABA-A, GABA-B and GABA-C receptors. The GABA-A receptor, whose amino acid sequence has been determined by cloning techniques is a pentameric structure composed of xcex1, xcex2, xcex3, xcex4 and/or xcfx81 subunits. So far, 6 xcex1 subunits, 3 xcex2 subunits, 3 xcex3 subunits, 1 xcex4 subunit and 2 xcfx81 subunits have been identified and sequenced. Five of these subunits (for example 2xcex11 2xcex22 xcex32) assemble to form a channel which is permeable to chloride ions. By binding to this GABA-A receptor, GABA increases the permeability of the channel to chloride ions, thus inhibiting neuronal transmission. In the light of the large number of possible permutations of the various subunits, a very high heterogeneity of the GABA-A receptor is observed in the brain of mammals and various structures of the brain generally show a preponderance for certain combinations of subunits.
The search for ligands selective for one of these various subunits of GABA-A receptors is a major objective of clinical medical research in this field.
Apart from GABA, a large number of various classes of compounds are known which bind to the GABA-A receptor. Some products, such as muscimol and isoguvacine, bind directly to the same site as GABA on the GABA-A receptor and stimulate the receptor in the same manner as GABA itself. Unlike these agonists, some substances, such as bicuculline (2), competitively inhibit the action of GABA. Such antagonists of the GABA receptor show convulsant properties in vivo (P. Krogsgaard-Larsen, B. Frolund, F. S. Jorgensen, A. Schousboe, J. Med. Chem., 1994, 37, 2489).
The inhibitory action of GABA may be modulated by compounds which interact with a variety of allosteric sites on the GABA-A receptor which are distinct from the GABA recognition site. One of the best known classes of allosteric modulators of the GABA-A receptor is that of the benzodiazepines (for example diazepam (3)). By thus binding to their own recognition site on the GABA-A receptor (the benzodiazepine receptor or BZR), these compounds improve the action of GABA by increasing the frequency of opening of the chloride channel (R. E. Study, J. L. Barker, Proc. Natl. Acad. Sci. USA, 1981, 78, 7180). This results in the anticonvulsant, anxiolytic, sedative-hypnotic and muscle-relaxing activities of these products which are widely used in the clinical field. Other classes of compounds which are structurally unrelated to the benzodiazepines, such as triazolopyridazines (for example Cl 218872 (4)), imidazopyridines (for example zolpidem (5)), cyclopyrrolones (for example zopicolone (6)) and xcex2-carbolines (for example xcex2-CCM (7)), can also bind to the benzodiazepine receptors. In the case of the latter, certain derivatives inhibit, rather than increase, the neuroinhibitory action of GABA (R. L. Macdonald, R. E. Twyman in xe2x80x9cIon Channelsxe2x80x9d ed. by T. Narahashi, Vol. 3, pp. 315-343, Plenum Press, New York, 1992). In this case, the compounds, which are generally convulsant, are called inverse agonists (or negative allosteric modulators) of BZR, to distinguish them from agonists (or positive allosteric modulators) of BZR which are therapeutically useful. Some of these products demonstrate selectivity on various subclasses of GABA-A/benzodiazepine receptors. Thus, zolpidem, which is clinically used as a hypnotic, is selective for the subclass of benzodiazepine receptors which are predominantly found in the cerebellum (BZ1 receptors) (S. Arbilla, H. Depoortere, P. George, S. Z. Langer, Naunyn-Schmiedeberg""s Arch. Pharmacol., 1985, 330, 248). This selectivity results in a narrower activity spectrum (for example anxiolysis without hypnotic effect) or in a reduction in the undesirable effects of this type of product (addiction, dependence, amnesia and the like).
Other sites exist on the GABA-A receptor which also make it possible, depending on its binding with an appropriate molecule, to modulate the activity of GABA. Among these sites, there may be mentioned those for neurosteroids (for example 3xcex1-OH-5xcex1-pregnan-20-one), barbiturates (for example pentobarbital), anesthetics (for example propofol), cage convulsants t-butyl-bicyclophosphorothionate which bind to the picrotoxin site of the GABA-A receptor (W. Sieghart, Pharmacol. Rev., 1995, 47, 181 and C. R. Gardner, W. R. Tully, C. J. R. Fiedgecock, Prog. Neurobiol., 1993, 40, 1). Other binding sites, which are less well characterized but which are apparently distinct, are those for loreclezole and xcex3-butyrolactones. Such compounds also positively modulate the action of GABA and this effect results in an anticonvulsant and/or anxiolytic action in vivo.
It is thus clear that a large number of allosteric modulatory sites, which can increase the action of GABA and thus demonstrate a therapeutic efficacy in a wide range of central nervous system disorders, exist on the GABA-A receptor. It can thus be reasonably concluded that novel chemical structures can discover other allosteric modulatory sites not yet characterized on the GABA-A receptor or bind to known sites with higher affinities or higher selectivities. Such compounds may, as a result, demonstrate a potent and/or highly specific activity as well as lower undesirable side effects in the treatment of such disorders. 
Wyeth laboratories claim that compound 17, which is obtained according to scheme 1 (and related compounds) are antagonists of the GABA autoreceptor and are thus useful for the treatment of central nervous system disorders and of pain (European patent No. EP0419247A2). The GABA autoreceptor is considered as being a pharmacological entity distinct from the GABA-A receptor itself. Type 17 compounds thus have no action or have a very weak action on GABA-A receptors. The GABA autoreceptor ligands exhibit different structural requirements from those of the GABA-A receptor. 
Bicuculline 2, whose molecular structure has been used as the starting point for designing the compounds of the present invention, is a natural product which was isolated from Dicentra cucullaria in 1932 by Manske (R. H. Manske, Can. J. Res., 1932, 7, 265). This compound, a potent convulsant, is a competitive antagonist of the GABA-A receptor which binds to the same site as GABA itself (M. A. Simmonds, Br. J. Pharmacol., 1978, 63, 495). Because of the convulsant nature of bicuculline, very few structure-function studies have been carried out on this molecule. Allan and Apostopoulos (R. D. Allan, C. Apostopoulos, Aust. J. Chem., 1990, 43, 1259) have described the preparation of bicuculline derivatives modified at the C-9 position starting from 9-hydroxymethylbicuculline whose synthesis has been described by Bhattacharyya et al. (A. Bhattacharyya, K. M. Madyastha, P. K. Bhattacharyya, M. S. Devanandan, Indian J. Biochem. Biophys., 1981, 18, 171). No biological data has been reported. Simonyi et al. (J. Kardos, G. Blasko, P. Kerekes, I. Kovacs, M. Simonyi, Biochem. Pharmacol., 1984, 33, 3537) have determined the GABA-A receptor activities of 45 phthalideisoquinoline alkaloids related to (+)-bicuculline. No compound was more active than bicuculline, but in all cases, the erythro stereoisomer (that is to say that of bicuculline) was more active than the threo isomer. This conclusion was confirmed by the same authors in related studies published in 1996 (J. Kardos, T. Blandl, N. D. Luyen, G. Dornyei, E. Gacs-Baitz, M. Simonyi, D. J. Cash, G. Blasko, C. Szantay, Eur. J. Med. Chem., 1996, 31, 761). The importance of the fused ring xe2x80x9cAxe2x80x9d of bicuculline for the GABA-A receptor activity has never been demonstrated, except in the present invention. While bicuculline was the starting point of our study, the most active molecules synthesized are considerably different from bicuculline in that:
the fused ring xe2x80x9cAxe2x80x9d is absent and the 3,4 bonds are saturated,
the threo rather than erythro isomers are the most active,
the aromatic benzo ring of the isoquinoline entity is not substituted,
the nitrogen atom of the isoquinoline entity is substituted with an aryl carbamate, an aryl sulfonate rather than a methyl group.
Finally, the compounds of the present invention bind at best only weakly to the GABA site on the GABA-A receptor (thus measured by H3-muscimol displacement studies) but rather bind to the benzodiazepine sites of the GABA-A receptor or to an as yet unidentified site of this receptor or, in some cases, to the latter two sites. More importantly, whereas bicuculline and these known analogs inhibit the currents produced by GABA in the neurons (giving rise to their pharmacological convulsant profile in vivo), the compounds of the present invention strongly stimulate the currents produced by GABA in the same manner as the therapeutically useful benzodiazepines.
The present invention therefore relates to the compounds of general formula: 
in which:
Z represents a sulfur atom, an oxygen atom, NH, N-alkyl or Nboc,
the groups Rxe2x80x2, which may be identical or different from one another, each represent a hydrogen atom, an OCH3 group or an OCH2O group,
the group Rxe2x80x3 represents a hydrogen or a CH3 group,
the group X represents a carbonyl, sulfonyl or CO2 group
and he group R represents an alkyl, aryl, alkenyl or aralkyl group
it being possible for these compounds to exist in the form of a racemic mixture or in an optically pure form.
A preferred group of compounds according to the invention consists of the compounds for which Z is an oxygen atom or NbOc. The compounds of the present invention for which Z is an oxygen atom are particularly preferred because of their high activity.
A particular group of compounds according to the invention consists of the compounds of general formula: 
in which:
the group X represents a carbonyl, sulfonyl or CO2 group,
and the group R represents an alkyl, aryl, alkenyl or aralkyl group.
Among the preferred compounds of the invention, there may be mentioned the compound of formula: 
and that of formula: 
The term alkyl group is understood to mean the linear or branched, substituted or unsubstituted alkyl groups having from 1 to 4 carbon atoms. Preferred examples of alkyl groups are the CH3 and C2H5 groups.
The term alkenyl group is understood to mean the linear or branched, substituted or unsubstituted alkenyl groups having from 1 to 4 carbon atoms.
The term aryl groups is understood to mean substituted or unsubstituted aromatic rings having from 5 to 8 carbon atoms, having one or more aromatic rings. The aromatic rings may be joined or fused. Examples of preferred aromatic rings are phenyls and naphthyls. Examples of preferred substituents of these rings are alkyl groups such as CH3, halogen atoms such as Cl, halogenated alkyl groups such as CF3 or groups of the OCH3 type.
The term aralkyl groups is understood to mean aryl groups, as defined above, linked to the sulfonyl or carbonyl or CO2 group via an alkyl group as defined above. A preferred example of an aralkyl group is the CH2Ph group.
All the compounds according to the invention possess an asymmetric center and can therefore exist in the form of optical isomers. The present invention comprises these isomers either separately or as a mixture.
The present invention also relates to the method of preparing these compounds which may be the following: the compound of general formula: 
in which:
Z represents a sulfur atom, an oxygen atom, NH, N-alkyl or Nboc, preferably an oxygen atom,
the groups Rxe2x80x2, which may be identical or different from one another, each represent a hydrogen atom, an OCH3 group or an OCH2O group,
the group Rxe2x80x3 represents a hydrogen or a CH3 group,
the group X represents a carbonyl, sulfonyl or CO2 group
and the group R represents an alkyl, aryl, alkenyl or aralkyl group,
is selectively hydrogenated with H2 in the presence of Pdxe2x80x94C to give the compounds according to the present invention.
This method may comprise a preliminary step of condensing, in the presence of an acylating or sulfonylating agent, a silyl enol ether represented by the general formula: 
in which:
Z represents a sulfur atom, an oxygen atom, NH, N-alkyl or Nboc, preferably an oxygen atom,
and Rxe2x80x3 represents a hydrogen atom or a CH3 with a salt of 3,4-dihydroisoquinoline represented by the following general formula: 
xe2x80x83in which:
the groups Rxe2x80x2, which may be identical or different from one another, each represent a hydrogen atom, an OCH3 group or an OCH2O group,
the group X represents a carbonyl, sulfonyl or CO2 group
and the group R represents an alkyl, aryl, alkenyl or aralkyl group.
The present invention also relates to pharmaceutical compositions comprising, as active ingredient, one of the compounds defined above and an appropriate excipient. These compositions may be formulated for administration to mammals, including humans. The dosage varies according to the treatment and according to the condition in question. These compositions are produced so as to be administrable by the digestive or parenteral route.
In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active ingredient may be administered in unit forms for administration, as a mixture with conventional pharmaceutical carriers, to animals or to human beings. The appropriate unit forms for administration comprise the forms for oral administration such as tablets, gelatin capsules, powders, granules and oral solutions or suspensions, forms for sublingual and buccal administration, forms for subcutaneous, intramuscular, intravenous, intranasal or intraocular administration and forms for rectal administration.
When a solid composition is prepared in the form of tablets, the principle active ingredient is mixed with a pharmaceutical vehicle such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic and the like. The tablets may be coated with sucrose or other appropriate materials or alternatively they may be treated such that they have a prolonged or delayed activity and such that they continuously release a predetermined quantity of active ingredient.
A preparation as gelatin capsules is obtained by mixing the active ingredient with a diluent and pouring the mixture obtained into soft or hard gelatin capsules.
A preparation in the form of a syrup or elixir may contain the active ingredient together with a sweetener, an antiseptic, as well as a flavoring agent and an appropriate coloring agent.
The water-dispersible powders or granules may contain the active ingredient as a mixture with dispersing agents or wetting agents, or suspending agents, as well as with flavor correctors or sweeteners.
For rectal administration, suppositories are used which are prepared with binders which melt at rectal temperature, for example cocoa butter or polyethylene glycols.
For parenteral, intranasal or intraocular administration, aqueous suspensions, isotonic saline solutions or sterile and injectable solutions are used which contain pharmacologically compatible dispersing agents and/or wetting agents.
The active ingredient may also be formulated in the form of microcapsules, optionally with one or more carrier additives.
The present invention also relates to the use of these compounds as stimulant of the activity of xcex3-aminobutyric acid acting via the GABA-A receptor of the central nervous system.
The compounds according to the invention and the pharmaceutical compositions comprising them may be used as a medicament, in particular for the treatment of nervous disorders. These disorders are preferably of the type including epilepsy, anxiety, depression, sleep and memory disorders, panic attacks, muscle contractions, pain, dependence on alcohol or on benzodlazepines or psychotic behavior.
The active compounds of the present invention (that is to say having the general structure 8) may all be prepared by the same method (scheme 2). Briefly, the substituted or unsubstituted derivatives of 3,4-dihydroisoquinoline 11 (which are prepared according to published methods: R. L. Hillard, C. A. Parnell, K. P. C. Vollhardt, Tetrahedron, 1983, 39, 905 and W. M. Whaley, M. Meadow, J. Chem. Soc., 1953, 1067) react first of all in acetonitrile at 0xc2x0 C. with an aromatic acid chloride or an aryl chloroformate or an arylsulfonyl chloride for 15 to 30 min. The reaction mixture is then treated with a dienolic silyl ether 10 (prepared by reacting commercial butyrolactone 9 with trialkylsilyl triflates according to published methods: (a) G. Casiraghi, L. Colombo, G. Rassu, R. Spanu, J. Org. Chem., 1990, 55, 2565, (b) Y. Morimoto, K. Nishida, Y. Hayashi, H. Shirahama, Tet. Lett., 1993, 34, 5773, (c) G. Casiraghi, G. Rassu, P. Spanu, L. Pinna, J. Org. Chem., 1992, 57, 3760, and (d) C. W. Jefford, A. W. Sledeski. J-C Rossier, J. Boukouvalas, Tet. Lett., 1990., 31, 5741). After 15 to 30 minutes, the reaction mixture is treated in order to give the unsaturated coupled product 12 in the form of a mixture of 4 isomers. The double bond of compound 12 is reduced by catalytic hydrogenation on palladium on carbon in ethanol to give the compounds having the general structure 8. The two diastereoisomers of 8 (erythro-8 and threo-8) may be separated by either a) silica gel chromatography, or b) fractional crystallization, or c) HPLC on a silica column. In the cases where, in compound 8, Z=N-Boc, the treatment with trifluoroacetic acid in dichloromethane gives the deprotected compound 8 (Z=NH). 
In the special case where the group xe2x80x9cRxe2x80x9d of compound 12 is a benzyloxycarbonyl group (xe2x80x9cCbzxe2x80x9d), then the erythro and threo isomers may be separated by silica gel chromatography. Catalytic hydrogenation of the compound threo-12 (the most active isomer) over palladium on carbon simultaneously leads to the reduction of the double bond and to the elimination of the Cbz group to give the compound threo-13. The reaction of the latter with an aromatic acid chloride or an aryl chloroformate or an arylsulfonyl chloride in the presence of triethylamine may then give the desired compound threo-8. The advantage of this second method (scheme 3) for preparing the compound threo-8 (or erythro-8) consists In the fact that it only requires a separation of the diastereoisomers of compound 12 (R=CO2CH2Ph) whereas in the first method, the diastereoisomers of each final product 8 should be separated (sometimes with difficulty). 
Finally, given that the erythro-8 and threo-8 compounds are always in a mixture of enantiomers, the latter may be suitably separated by HPLC on a column with a chiral support.
The compounds synthesized are tested in vitro for their capacity to bind to various sites on the GABA-A receptor, including the site for GABA itself (by H3-muscimol displacement studies), the site for benzodiazepines (by displacement of H3-flunitrazepam), the site for picrotoxin (by displacement of 35S-TBPS). Briefly, frozen membranes obtained from cerebellum or cerebellum-free brain are thawed, centrifuged and resuspended in 50 mM Tris-citrate buffer, pH 7.4, at a protein concentration of about 1 mg/ml. The membranes (0.5 ml) are then incubated in a total of 1 ml of solution containing 50 mM Tris-citrate buffer, pH 7.4, 150 mM NaCl and 2 nM [3H]flunitrazepam or 2 nM [3H]muscimol in the absence or in the presence of varying concentrations of the compound to be studied or of 10 xcexcM diazepam or of 10 xcexcM GABA, for 90 min at 4xc2x0 C., respectively. For the [35S]TBPS binding, the membranes are incubated in a total of 1 ml of solution containing 50 mM Tris-citrate buffer, pH 7.4, 200 mM NaBr and 2 nM [35S]TBPS in the absence or in the presence of varying concentrations of the compound to be studied or of 10 xcexcM TBPS or picrotoxinin for 180 min at room temperature (W. Sieghart, A. Schuster, Biochem. Pharmacol., 1984, 33, 4033, and J. Zezula et al., Eur. J. Pharmacol., 1996, 301, 207).
The membranes are then filtered through Whatman GF/B filters. When the binding of [3H]flunitrazepam or of [3H]muscimol is studied, the filters are rinsed twice with 5 ml of a buffer solution of 50 mM ice-cold Tris-citrate. When the binding of [35S]TBPS is studied, the filters are rinsed three times with 3.5 ml of this buffer solution. The filters are transferred into scintillation flasks and subjected to scintillation counting after adding 3.5 ml of scintillation fluid. The nonspecific binding, determined in the presence of 10 xcexcM diazepam, 10 xcexcM GABA or 10 xcexcM TBPS, is subtracted from the total of the binding of [3H]flunitrazepam, of [3H]muscimol or of [35S]TBPS, respectively, in order to obtain the specific binding.
The compounds synthesized are also studied for their capacity Lo cause the opening of the GABA-A receptor channel or to allosterically modulate the currents caused by GABA. For this purpose, the recombinant GABA-A receptors are expressed in Xenopus oocytes. Briefly, the Xenopus laevis oocytes are prepared, injected, defolliculated and the currents are recorded in the manner described (E. Sigel, J. Physiol., 1987, 386, 73 and E. Sigel, R. Baur, G. Trube, H. Mxc3x6hler, P. Malherbe, Neuron, 1990, 5, 703). The oocytes are injected with 50 nl of cRNA dissolved in 5 mM K-Hepes (pH 6.8). This solution contains the transcripts encoding the various subunits at a concentration of 10 nM for xcex11, 10 nM for xcex22 and 50 nM for xcex32. The RNA transcripts are synthesized from linearized plasmids encoding he desired protein using the message machine kit (Ambion) according to the manufacturers"" recommendation. A poly(A) tail of about 300 residues is added to the transcripts using the yeast poly(A) polymerase (USB or Amersham). The cRNA combinations are coprecipitated in ethanol and stored at xe2x88x9220xc2x0 C. The transcripts are quantified on agarose gels after staining with Radiant Red RNA stain (Bio-Rad) by comparing the staining intensities with various quantities of molecular weight markers (RNA-Ladder, Gibco-BRL). The electrophysiological experiments are carried out by the two-electrode voltage clamp method at a holding potential of xe2x88x9280 mV. The medium contains 90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2 and 10 mM Na-Hepes (pH 7.4). GABA is applied for 20 s and a washing period of 4 min is allowed to ensure complete restoration of desensitization. The perfusion system is cleaned between applications of the compounds by washing with dimethyl sulfoxide in order to avoid contamination. The compounds are applied at a concentration of 100 microM in the absence of GABA to see if they can act as channel agonists. To study allosteric modulation, GABA is first of all applied alone and then in combination with either 0.1 microM or 100 microM of compounds.
The compounds synthesized are tested in vitro for their capacity to displace tritiated flunitrazepam (3H-Flu, a ligated selective for the benzodiazepine-binding site of the GABA-A receptor), 35S-TBPS (selective for the picrotoxin-binding site) and tritiated muscimol (selective for the GABA-binding site). These compounds are also tested for their capacity to prevent or to stimulate the currents caused by GABA in the oocytes of frogs expressing the xcex11xcex22xcex32 subtype of the GABA receptor.
The detailed results for examples of compounds are shown in tables 1a to 1i.
The most active compounds are summarized in the following table 2.
These examples of compounds and the results obtained with them are indicated without limitation and illustrate the invention.
The conclusions which may be drawn from the study of structure-function are the following:
The most active compounds (that is to say those producing the highest stimulation of the currents produced by GABA) have:
1xe2x80x94a 3xe2x80x2,4xe2x80x2 unsaturated bond (compare compounds 54 and 50);
2xe2x80x94an oxygen atom at the 1xe2x80x2 position rather than a nitrogen atom (compare compounds 67 and 73);
3xe2x80x94the hydrogens at the C-1 and C-2xe2x80x2 position have a relative xe2x80x9cthreoxe2x80x9d rather than xe2x80x9cerythroxe2x80x9d configuration (that is to say of the bicuculline type) (compare compounds 48 and 49);
4xe2x80x94a positive rather than a negative optical rotation in the case of the pure enantiomers of the xe2x80x9cthreoxe2x80x9d diastereoisomers (compare compounds 69 and 68);
5xe2x80x94no alkyl ether substituent on the xe2x80x9cAxe2x80x9d ring (compare compounds 54 and 48);
6xe2x80x94an aryl carbamate or an aryl sulfonate (that is to say the group R) attached to the nitrogen atom of isoquinoline (compare 48 and 69 with 55).
As regards the mechanism by which these novel compounds stimulate the current produced by GABA, several conclusions may be drawn from the studies of radioactive ligand displacement combined with the electrophysiological results. First of all, none of these compounds binds with the GABA recognition site of the GABA-A receptor. Thus, no, or a very weak displacement of tritiated muscimol is observed in the presence of the compounds of table 2. Furthermore, in the absence of GABA, no current is produced in the oocyte preparations when any one of these compounds is administered.
The compounds in which the group xe2x80x9cRxe2x80x9d is a carbamate (for example 54) or another substituent Nxe2x80x94Cxe2x95x90O (for example 59, 61) appear to stimulate the GABA currents by binding with the benzodiazepine recognition site of the GABA-A receptor in a manner similar to benzodiazepines themselves (diazepam for example). Thus, compound 54 displaces tritiated flunitrazepam with an IC50 of 40 nM for the type of receptors found in the cerebellum (BZ1 subtype) and 150 nM for the receptors of the remaining part of the brain (BZ2 receptor subtype). These values compare favorably with those for diazepam, the benzodiazepine prototype, which, nevertheless, demonstrates no selectivity on the subtypes relative to the novel compounds such as compound 54. The potent stimulation of the GABA currents which is produced by the compounds of the 54 type may be prevented to a large extent by the addition of all antagonist of the benzodiazepine receptor in the preparation of oocytes. Thus, 78% of the stimulation produced by 10 xcexcM of compound 54 is prevented by the coadministration of 1 xcexcM flumazenil. This proves that the binding of compound 54 (and of similar analogs) to the benzodiazepine receptor is widely responsible for the stimulation of the GABA currents which is observed.
A remarkable effect is observed with the molecules in which the group R is an arylsulfonyl (for example compounds 65-69). In all cases, the stimulations of the current are much more vigorous than with the analogous carbamate derivative 54. However, the binding affinities with the benzodiazepine receptor are considerably lower. Thus, compound 69 stimulates the GABA current 2.5-fold more strongly than compound 54, and yet shows a binding affinity 250-fold lower for the benzodiazepine receptor. Furthermore, only 28% of the stimulant activity of compound 69 is prevented by flumazenil, an antagonist of BZR. These results clearly indicate that, by far, the largest portion ( less than 70%) of the potent stimulatory activity of GABA produced by the arylsulfonyl derivatives such as compound 69 is due to their interaction with a binding site distinct from those for the benzodiazepines. Although the arylsulfonyl derivatives equally weakly displace TBPS from its binding site on the GABA-A receptor, there is no observable correlation between these binding affinities and the stimulant activities of GABA (table 2).
Other experiments demonstrate that although compound 69 does not produce currents by itself, it can increase the currents produced by pentobarbital. By contrast, the currents produced by GABA which are stimulated by pentobarbital are not further stimulated by compound 69. Furthermore, a mutated receptor incapable of responding to an application of loreclezole shows an unimpaired stimulation of the current by compound 69. Finally, the recombinant receptors of the composition of xcex1xxcex22xcex32 subunits in which x=1, 2, 3, 5, 6 are also tested for the stimulation by compound 69 of the GABA current. The highest stimulation of the current is observed for x=6.
It can therefore be concluded that whereas the carbamate compounds such as 54 represent a novel and a selective subclass of ligands which are agonists of the benzodiazepine receptor, the arylsulfonyl analogs, although demonstrating the same type of GABA stimulating activity as the benzodiazepines, only do so by banding mainly to an as yet noncharacterized site of the GABA-A receptor. These compounds are therefore novel both from a structural point of view and also from the point of view of their novel mode of action. The GABA-stimulating activities of these compounds have a therapeutic benefit, which is demonstrated by the fact that benzodiazepines are widely prescribed. The apparent novel mode of action of the compounds of the present invention suggests that they can possess the beneficial clinical effects of benzodiazepines but with weaker or even no side effects.
It was surprisingly noted that compounds possessing a double bond in the isoquinoline part (compounds 75 and 76) are particularly active.
Furthermore, some of these products demonstrate selectivity for one or other of the numerous GABA-A receptor subtypes. Thus, although compound 32a (table 1b) only weakly stimulates the type xcex11xcex22xcex32 receptor, this stimulation is greatly increased in cases where the xcex11 subunit is replaced by the xcex12, xcex13, xcex15 or xcex16 subunit. Likewise, compound 73 (table 1g) only weakly stimulates the xcex11xcex22xcex32 receptor but in the presence of receptors possessing an xcex15 subunit, an inhibition (instead of a stimulation) of the gabaergic currents is observed. This result demonstrates that 73 as well as the other analogous molecules of the present invention have a beneficial effect on memory.